Myogenesis is an in vivo process occurring during embryogenesis and tissue repair. Satellite cells begin to proliferate and form myoblasts. A myoblast is a mononucleate cell type that, by fusion with other myoblasts (this is a part of a process being also called “differentiation”), gives rise to myotubes that maturate and later eventually develop into muscle fibers. Maturation of myotubes can easily be characterized through the striated organization of myosin heavy chain molecules within sarcomeres, and the location of nuclei at the center of the structure [Abmayr 2012]. When myotubes are grown in a cell culture device, the growth method is intended to influence the growth of myotubes that are as close as possible as physiological myotubes.
Instead of substrates having a uniform, unpatterned cell-adhesive surface that generally provide a random orientation of the myotubes, patterned substrates have been demonstrated to enhance a specific orientation of myotubes.
Said patterns are typically in the form of lines, although finite geometrical shapes such as circular, square and Y-shaped patterns have also been investigated [Junkin 2011].
These experiments show that myotube morphological properties are strongly influenced by the shape of the pattern on which they are grown.
Other patterns consist of linear grooves formed in the surface of a substrate by an etching technique [Yamamoto 2008].
These lines are considered of an “infinite” length, meaning that their length is much greater than the length of the myotubes.
Some variations of these linear patterns have been tested in order to define the most appropriate shape with regard to myotube orientation.
In particular, so-called “hybrid” patterns that consist of the combination of a linear element and an arcuate element centered on the linear element, have proven to increase both the fusion index, i.e. the ratio of the number of nuclei in myocytes having two or more nuclei versus the total number of nuclei, and the maturation index, i.e. the number of myotubes having five or more nuclei; in addition, these pattern also provided a good alignment of the myotubes [Bajaj 2011]. More precisely, the pattern that provide the best results in this respect consists of a linear element having a width of 100 μm and a length of 2000 μm, whereas the arc degree of the arcuate element is of 30°. This so-called “hybrid” pattern is illustrated in FIG. 1.
Myoblast patterning on soft substrate (Young's modulus close to 10 to 15 kPa) have been shown to provide oriented myotubes with an increased maturity [Engler 2004].
However, although myotube orientation is extensively studied in the literature, their morphological parameters are poorly quantified and highly variable, due to difficulties in individualizing full mature structures and standardizing their morphology. This high variability level does not permit robust cell based assay development. Also, the level of achieved myotube maturation is still fare from in vivo like in term of striation and nuclei location.